Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer.
Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer.
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DOI：
10.1186/bcr1874
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发表时间：
2008
影响因子：
7.4
通讯作者：
Samant, Rajeev S.
中科院分区：
文献类型：
--
作者：
Mitra, Aparna;Fillmore, Rebecca A.;Metge, Brandon J.;Rajesh, Mathur;Xi, Yaguang;King, Judy;Ju, Jingfang;Pannell, Lewis;Shevde, Lalita A.;Samant, Rajeev S.
DnaJ的哺乳动物近亲（MRJ [DNAJB6]）是人类DnaJ家族的新成员，具有两个同工型。较小的异构体MRJ(S)主要因其在亨廷顿病中的可能作用而被研究。没有任何关于长亚型MRJ(L)生物活性的报道。我们调查了这种分子是否在乳腺癌中起作用。我们对乳腺癌细胞系和乳腺癌组织微阵列中MRJ的表达进行了有趣的观察，如下所述，这促使了我们的研究。采用实时荧光定量PCR技术检测了几种乳腺癌细胞系中MRJ(L)的表达。用Western blot方法研究了乳腺癌细胞系中大、小亚型的相对水平。采用抗mrj抗体对乳腺癌进展组织芯片进行探针检测。MRJ(L)在两种乳腺癌细胞系中异位表达。这些细胞系被评估其肿瘤侵袭性的体外相关性，如侵袭、迁移和锚定独立性。我们还评估了这些细胞系的体内肿瘤生长和转移情况。通过分析MRJ(L)表达体分泌的蛋白质组，阐明MRJ(L)重新表达后所带来的生化变化。我们发现，与正常乳腺相比，MRJ(L)在侵袭性乳腺癌细胞系中的表达水平显著降低。此外，在乳腺癌的临床病例中，MRJ的表达随着浸润性导管癌的分级而消失。重要的是，在那些也有淋巴结转移的病例中，MRJ染色丢失。我们报道了MRJ(L)是一个具有功能性核定位序列的蛋白。在乳腺癌细胞系MDA-MB-231和MDA-MB-435（一种从乳腺脂肪垫转移的细胞系）中，通过外源性启动子表达MRJ(L)可以减少它们的迁移和侵袭，降低它们的运动性，并显著降低裸鼠原位肿瘤的生长。此外，MRJ(L)表达细胞的分泌蛋白组显示肿瘤进展和转移促进分泌蛋白水平降低，如SPP1（骨桥蛋白）、AZGP1（锌结合α2-糖蛋白1）、SPARC（骨连接蛋白）、NPM1（核磷蛋白）和VGF （VGF神经生长因子诱导）。另一方面，MRJ(L)表达细胞的分泌蛋白组中分泌的转移抑制因子KiSS1（黑色素瘤转移抑制因子）水平升高。我们通过定量RT-PCR分析证实，分泌谱反映了相应基因的转录改变。总的来说，我们的数据表明，DNAJB6的一个完全未表征的亚型在乳腺癌中起重要作用。我们发现MRJ(L)是一种在乳腺癌中缺失的核蛋白，它调节了肿瘤形成和转移的几个关键因素，并且在功能上能够延缓肿瘤的生长。
Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below. Expression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L). We found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding α2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes. Collectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.
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